Use of anabolic agents, anti-catabolic agents, antioxidant agents, and analgesics for protection, treatment and repair of connective tissues in humans and animals

ABSTRACT

The present invention relates to compositions for the protection, treatment and repair of connective tissues in humans and animals comprising any or all of anabolic, anti-catabolic, anti-oxidant and analgesic agents, including aminosugars, S-adenosylmethionine, arachadonic acid, GAGs, including pentosan, collagen type II, tetracyclines or tetracycline-like compounds, diacerin, super oxide dismutase, L-ergothionine, one or more avocado/soybean unsaponifiables, and an analgesic, e.g., acetaminophen, and to methods of treating humans and animals by administration of these novel compositions to humans and animals in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

In connection with this application, priority is claimed to thefollowing provisional application, A COMPOSITION OF ACETAMINOPHEN, ANAMINOSUGAR AND A GLYCOSAMINOGLYCAN, U.S. Ser. No. 60/088,205, filed Jun.5, 1998. The present application is also a continuation-in-partapplication of U.S. patent application Ser. No. 09/249,335, filed Feb.12, 1999, the disclosure of which is hereby incorporated by referenceherein in its entirety. That application claimed priority to provisionalapplication: THE USE OF ANABOLIC AGENTS, ANTI-CATABOLIC AGENTS,ANTIOXIDANT AGENTS, AND ANALGESICS FOR PROTECTION, TREATMENT AND REPAIROF CONNECTIVE TISSUES IN HUMANS AND ANIMALS, U.S. Ser. No. 60/074,594,filed Feb. 13, 1998.

FIELD OF THE INVENTION

The present invention relates to compositions for the protection,treatment and repair of connective tissues in humans and other animals.

BACKGROUND OF THE INVENTION

The tissues of mammals, including humans, are in a constant state offlux between the anabolic processes that build up tissues, and thecatabolic processes which degrade tissues. The state of health existswhen there is a balance between these two processes, and derangements ofthe balance produce disease. This holds true for all tissues of thebody. Connective tissues are of particular importance for severalreasons. First, they support the “functional cells” of the body, i.e.,epithelial, muscle and neural cells. Second, they play critical roles inintercellular communication, which is essential for multicellular life.

The inflammatory process occupies a key position in this balance. Wheninjury to tissues occurs, inflammation initiates the biochemicalprocesses that result in tissue repair. Because inflammation results inthe symptoms of pain, inflammation, and swelling of the tissuesinvolved, it is often regarded by both patients and physicians as anabnormal and undesirable state, which should be treated and relieved assoon and as completely as possible. As a result, pharmacies are full of“anti-inflammatory drugs” (such as corticosteroids and the non-steroidalanti-inflammatory drugs, such as aspirin). Under certain circumstances,inflammation can indeed be destructive; however, it is important toremember that inflammation is closely linked with tissue healing.Indeed, inflammation is not easily categorized as strictly anabolic orcatabolic—it may have either effect. Its purpose in the body is toremove, dilute or wall-off the injurious agent(s). It also sets intomotion the biochemical processes that repair and reconstruct the damagedtissue. Because it is essential to healing, and because it can alsocause tissue destruction, inflammation and its mediators are importantfactors in the anabolic and catabolic balance.

One very important class of inflammatory mediators is the eicosanoidgroup. The eicosanoids are synthesized in the body from essential fattyacids (“FAs”). Through a series of biochemical reactions, the precursorfatty acids are modified to produce intermediate metabolites,arachadonic acid (“AA”), an omega-6 FA; and eicosapentanoic acid(“EPA”), an omega-3 FA. Eicosanoids produced from arachidonic acidinclude the 2-series of prostaglandins and the 4-series of leukotrienes,which are generally proinflammatory. The eicosanoids derived from EPA,such as the 3 series prostaglandins and hydroxyeicosapentaenoic acid(“HEPE”), are less inflammatory than those derived from AA. In addition,such eicosanoids may even have anti-inflammatory effects.

As a class, the eicosanoids are short-lived and locally active. They areresponsible for the initial events of inflammation, includingvasodilation, increased vascular permeability, and chemotaxis. Moreover,the eicosanoids are instrumental in the early steps of the healingprocess. For example, the eicosanoids trigger the release of cytokinessuch as TGF-B, which in turn stimulates the migration and proliferationof connective tissue cells, and the deposition of extracellular matrix.Specific constitutive eicosanoids also have protective effects in thegastrointestinal mucosa and kidney, because they maintainglycosaminoglycan synthesis and normal perfusion of these organs.

Because of anabolic processes such as these, and because of theinfluence of natural anti-catabolic and anti-oxidant agents in the body,the outcome of the majority of cases of inflammation is resolution ofthe injury and healing of the damaged tissues. Only in pathologicsituations does inflammation itself become a contributor to disease.

Research on the therapeutic use of eicosanoid precursor FAs (includingcis-linoleic and alpha-linolenic acids, the so-called omega-3 andomega-6 fatty acids) has been primarily directed towards their use ascompetitive inhibitors of the synthesis of eicosanoids, and therefore,their anti-inflammatory effects. Except in cases of severe or absolutedietary deficiency, little attention has been given to the beneficial,anabolic effects that the eicosanoids have in connective tissues.However, naturally occurring “subclinical” deficiencies of eicosanoidsprobably contribute significantly to disease, and are under diagnosed.For example, the enzyme delta-6-desaturase is responsible for thecommitted step in the synthesis of AA. Activity of this enzyme,(delta-6-desaturase) decreases with age. This is likely to prove asignificant factor in the increased incidence of connective tissuedysfunction in older population segments since a deficiency of AA woulddecrease anabolic processes and allow catabolic events to dominate.

Given the importance of inflammation in the healing of tissues, and theprotective role that some eicosanoids play, it is not surprising thatpharmaceuticals that decrease inflammation by blocking eicosanoidproduction should also have negative effects on healing and anabolicprocesses. It has long been known that corticosteroid drugs, which arestrongly anti-inflammatory, also delay healing and decrease theproduction of extracellular matrix components. This is because cortisoland related compounds stabilize cell membranes and therefore inhibit therelease of phospholipase A2, the precursor of AA. Recently attention hasturned to the non-steroidal anti-inflammatory drugs (“NSAIDs”). Numerousstudies have shown that NSAIDs, like corticosteroids, can decrease thesynthesis of matrix components by connective tissue cells, because theyinhibit prostaglandin endoperoxide synthase, and thus block thecyclooxygenase pathway.

Since the inflammatory process is the sine qua non of tissue healing,and since the eicosanoids are the mediators of the inflammatory process,the use of AA (and other eicosanoid compounds) is a novel approach totherapy of injured tissues. Kirkpatrick et al. investigated the use ofprostanoid precursors on chick embryonic cartilage in organ culture andfound no significant effects. [Kirkpatrick, C. J., “Effects ofProstanoid Precursors and Indomethacin on Chick Embryonic CartilageGrowth in Organ Culture,” Expl. Cell Biol., 51:192-200 (1993)]. Theexperimental model in this work may have contributed to the absence ofsignificant effects, because avian cartilage and embryonic cartilagediffer significantly from mammalian, postnatal cartilage. For example,embryonic cartilage of any species is hypermetabolic and anabolic tobegin with because it is in a period of exponential growth. Kent et al.examined the effects of AA in lapine cartilage and found a positiveeffect, although previous and subsequent research failed to confirmthis. [Kent, L. et al., “Differential Response of Articular ChondrocytePopulations to Thromboxane B2 and Analogs of Prostaglandin CyclicEndoperoxidases,” Prostaglandins, 19:391-406 (1980)]. Kirkpatrick andGardner found that AA and various metabolites of AA had insignificant orinhibitory effects on biosynthesis. [Kirkpatrick C. J. and Gardner, D.L., “Influence of PGA1 on Cartilage Growth,” Experientia, 33(4):504(1976)]. Lippiello, et al. found, however, that AA and other omega-6fatty acids had beneficial effects on chondrocyte metabolism in cellculture. [Lippiello, L., Ward, M., “Modification of articular cartilagechondrocyte metabolism by in vitro enrichment with fatty acids(abstract),” Trans. Orthop. Res. Soc. 13:162 (1988); Lippiello, L.,“Prostaglandins and articular cartilage; does Prostaglandin perturbationperpetuate cartilage destruction?” Semin Arthritis Rheum 11:87 (1981).]These variable results are not unexpected, since the balance betweenanabolic and catabolic processes in the body is delicate and easilyperturbed. Phan et al., suggest that products of AA via thecyclooxygenase pathway are anti-fibrogenic while AA products via thelipoxygenase pathway are pro-fibrogenic. This phenomenon demonstratesthe complexity of the eicosanoids' interactions.

Catabolic events are typically mediated in the body by enzymes thatbreak apart body constituents. Catabolism is essential for health anddeficiency of necessary enzymes results in disease, such as theso-called storage diseases like mucopolysaccharhidosis. Excessivecatabolism may also result in the breakdown of tissues and lead todisease, as in degenerative diseases like osteoarthritis or autoimmunediseases like multiple sclerosis. Various anti-catabolic substances inthe body help contain and balance catabolism. For example, chondroitinsulfate counteracts metalloproteinases that catabolize collagen andproteoglycans in the cartilage matrix. Similarly, alpha-one anti-trypsininhibits the effects of elastase, which contributes to alveolarbreakdown in emphysema.

Oxidative damage also has an impact on the balance of anabolism andcatabolism in the body. This damage is the result of the effects of freeradicals, substances that have an unpaired electron. Free radicals formconstantly in the body as the result of normal reactions like theproduction of ATP. They also form during the inflammatory process. Freeradicals cause cellular damage because they are highly chemicallyreactive. Because they have only a single electron, (a condition thatnature abhors as it does a vacuum), these substances “steal” electronsfrom molecules in their vicinity. The molecules making up cellstructures, such as the cell membrane or DNA are thereby renderedelectron-deficient. The deficiency of electrons in turn makes the cellstructure unstable and cell dysfunction occurs, including manufacture ofabnormal proteins, cell rupture, and cell death. Oxidative damage isimplicated in many catabolic events in the body, including the agingprocess. Anti-oxidants, such as vitamin. C, vitamin E, superoxidedismutase (SOD), selenium, and glutathione are substances that scavengefree radicals before oxidative damage occurs. In the sense that theyprevent cell damage, anti-oxidants are a specific type of anti-catabolicagent.

The body also contains anabolic compounds that stimulate tissue growth.Glucosamine is an amino sugar naturally formed in the body from glucose.When supplied exogenously, glucosamine stimulates connective tissue cellsynthesis, and thereby increases the amounts of normal extracellularmatrix. Glucosamine is also the building block for glycosaminoglycans incartilage and other connective tissues. Supplying additional glucosaminethus supplies the body with extra raw materials for matrix synthesis inconnective tissues. Other examples of anabolic compounds in the bodyinclude somatotropin, which stimulates protein synthesis, and thesomatomedins or insulin-like growth factors, which stimulate theproliferation of chondrocytes and fibroblasts and enhance matrixsynthesis.

The actions and interactions of these compounds are complex. A givencompound may have different effects in different tissues. For example,somatotropin increases protein synthesis (anabolism), but also speedsfat breakdown (catabolism). The effects that a particular compound orcombination of compounds will have depend on many factors, includingroute of administration, dosage, and duration of therapy.

Previous researchers have investigated the use of individual compoundsfor their anabolic, anti-oxidant or anti-catabolic effects. Glucosaminehas been found in cell culture to stimulate connective tissue cells toproduce the components of the matrix: collagen and glycosaminoglycans(GAGs). [Jimenez, S., “The Effects of Glucosamine sulfate on ChondrocyteGene Expression,” Eular Symposium, Madrid October 1996 Proceedings, page8-10]. S-adenosylmethionine is known to participate in several synthesisreactions, including the sulfation of GAGs. [Champe, P. Biochemistry,2^(nd) edition, J.B. Lippincott Co, Philadelphia, 1994, pp. 248, 250,265]. Arachadonic acid has been found to stimulate corneal healing.[Nakamura, M., “Arachidonic Acid Stimulates Corneal EpithelialMigration”, J. Ocul. Pharmocol., Summer: 10(2): 453-9 (1994)]. Thesecompounds therefore have anabolic effects.

Chondroitin sulfate has been shown to inhibit degradative enzymes,including the metalloproteinases that destroy cartilage matrix.[Bartolucci, C., “Chondroprotective action of chondroitin sulfate,” Int.J. Tiss. Reac., XIII(6):311-317 (1991)]. Studies with pentosan sulfatehave shown that it prevents complement-mediated damage in a rabbitmyocardial cells. [Kilgore, K., “The Semisynthetic PolysaccharidePentosan Polysulfate Prevents Complement-Mediated Myocardial Injury inthe Rabbit Perfused Heart,” J. Pharmocol. Exp. Ther., 285(3):987-94(1998)]. Oral administration of collagen type II has been shown todecrease the deleterious immune response that destroys joint tissue inrheumatoid arthritis. Tetracycline analogues are potent inhibitors ofmatrix metalloproteinases. [Ryan, M., “Potential of Tetracyclines toModify Cartilage Breakdown in Osteoarthritis.” [Curr. Opin. Rheumatol.,8(3): 238-47 (1996)]. Diacerein modifies the inflammatory process byinhibiting interleukin-1 activity, and also by direct effects onlymphocytes and neutrophils. [Beccerica, E., “Diacetylrhein and rhein:in vivo and in vitro effect on lymphocyte membrane fluidity,” Pharmocol.Res., 22(3):277-85 (1990); Mian, M., “Experimental Studies onDiacerhein: Effects on the Phagocytosis of Neutrophil Cells fromSubcutaneous Carregeenan-Induced Exudate,” Drugs Exp. Clin. Res.,13(11):695-8 (1987); Spencer, C., “Diacerein”, Drugs, 53(1):98-106(1997)]. These compounds can be classed as anti-catabolic agents.

L-ergothionine scavenges hydroxyl radicals and may inhibit singletoxygen formation, [Han JS. “Effects of Various Chemical Compounds onSpontaneous and Hydrogen Peroxide Induced Reversion in Strain TA104 ofSalmonella typhimurium,” Mutant Res., 266(2):77-84 (1992)], whilesuperoxide dismutase scavenges superoxide radicals [Mathews C.,Biochemistry 2^(nd) ed., Benjamin/Cummings Pub. Co., Menlo Park Calif.,1996, page 551]. These compounds can be classified as anti-oxidants.

Although these compounds have been investigated individually, to ourknowledge no one other than the present inventors has examined theeffects of certain combinations of any or all of anabolic,anti-catabolic and anti-oxidant agents to maintain health and to promotehealing. According to the present invention, combinations of theseagents can be used to maximize appropriate anabolic effects (healing)and decrease undesirable catabolic effects (degradation) and oxidativedamage, while at the same time, causing minimal or no adverse reactions.Therefore, it can be seen that there exists a need to providecompositions that will make use of the beneficial effects ofcombinations of anabolic agents, anti-catabolic agents, anti-oxidantand/or analgesic agents for the maintenance and repair of connectivetissues in humans and animals.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods oftreating repairing, and preventing damage to connective tissues inhumans and animals using such compositions. Therefore, it is an objectof the invention to provide novel compositions of any or all ofanabolic, anti-catabolic, anti-oxidant and/or analgesic agents for theprotection, treatment and repair of connective tissues in humans andanimals.

It is another object of the present invention to provide methods oftreating and repairing connective tissue in humans and animals withcompositions containing any or all of anabolic, anti-catabolic,anti-oxidant and/or analgesic agents.

It is still another object of the present invention to providecompositions any or all of anabolic, anti-catabolic, anti-oxidant and/oranalgesic agents selected from the group consisting of aminosugar,S-adenosylmethionine (SAMe), arachadonic acid (AA), GAG, pentosansulfate, collagen type II, tetracyclines, diacerin, super oxidedismutase (SOD), L-ergothionine, one or more avocado/soybeanunsaponifiables (ASUs) and analgesics, such as acetaminophen.

It is a further object of the present invention to provide compositionsto repair, treat, and prevent damage to connective tissue in humans andanimals that contain one or more of the elements selected from the groupconsisting of aminosugar, SAMe, arachodonic acid, GAG, pentosan sulfate,collagen type II, tetracyclines, diacerin, SOD, L-ergothionine, one ormore ASUs and analgesics, e.g, acetaminophen.

These and other objects of the present invention are apparent from thedetailed description and claims below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a detailed description of the biosynthetic pathway forthe creation of GAGs such as chondroitin sulfate.

FIG. 2 is the molecular structure of SAMe and its immediate precursor.

FIG. 3 provides a simplified diagram of the function of SOD.

FIG. 4 provides some examples of unsaponifiable lipids.

FIG. 5 is the molecular structure of acetaminophen.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention, used to treat, repair, andprevent damage to connective tissue, include combinations of anabolic,anti-catabolic, and/or anti-oxidant agents. Ingredients of preferredembodiments include compositions selected from the group consisting ofaminosugars, SAMe, AA, GAGs, including pentosan, collagen type II,tetracyclines, diacerin, SOD, L-ergothionine, and one or more ASUs.Optionally, the combinations of the present invention also include oneor more analgesics, such as acetaminophen. In addition, the presentinvention covers methods of administering these novel compositions tohumans and animals in need thereof.

Glucosamine—an example of an aminosugar—is naturally formed in the bodyfrom glucose. When supplied exogenously, glucosamine stimulatesconnective tissue cell synthesis, increasing the amounts of normalextracellular matrix. Glucosamine is also the building block forglycosaminoglycans (“GAGs”) in cartilage and other connective tissues,thus, supplying additional glucosamine supplies the body with extra rawmaterials for matrix synthesis in connective tissues. The aminosugarcomponent of the compositions of the present invention may comprisenatural, synthetic or semi-synthetic aminosugars including but notlimited to salts of glucosamine including glucosamine hydrochloride andglucosamine sulfate, glucosamine phosphate, and N-acetylglucosamine andsalts and/or mixtures thereof. In addition, the term aminosugar is alsoused herein to encompass aminosugars that may have been chemicallymodified yet retain their function. Such chemical modifications includebut are not limited to esterification, sulfation, polysulfation,acetylation, and methylation. Moreover, it is contemplated that the termaminosugar can extend to any composition of matter that isinsubstantially different from the aminosugar as above-described.

The GAG component of the compositions of the present invention maycomprise natural, synthetic or semisynthetic GAGs, GAG-like compounds,or GAG precursors, including but not limited to chondroitin, hyaluronicacid, glucuronic acid, iduronic acid, keratan sulfate, heparan sulfate,dermatin sulfate, and fragments, salts, and mixtures thereof. Inaddition, the term GAG as used herein further encompasses GAGs that havebeen chemically altered yet retain their function. Such modificationsinclude but are not limited to esterification, sulfation, polysulfation,and methylation. In fact, sulfated GAGs are a preferred component of thecompositions of the present invention. Hence, mono-sulfated andpolysulfated (or oversulfated) GAGs are preferred GAG components of thecompositions of the present invention. The term GAGs also is intended toencompass alternative nomenclature for the same group of above-describedcompounds—e.g., mucopolysaccharides, proteoglycans, and heparanoids. Inaddition, the GAG or GAG-like component of the compositions of thepresent invention may be derived from plant or animal sources, includingbut not limited to beechwood tree, to forms of animal cartilageincluding shark cartilage, bovine trachea, whale septum, and porcinenostrils, and to invertebrates such as Perna canaliculus and seacucumber.

Chondroitin sulfate is a preferred GAG. Chondroitin sulfate is the mostabundant glycosaminoglycan in articular cartilage and is also present inmany other connective tissues in the body. Additionally, chondroitinsulfate competitively inhibits degradative enzymes that degradeconnective tissues under conditions of abnormal, excessive inflammation.Chondroitin sulfate is a polymer composed of repeating units ofglucuronic acid and sulfated galactosamine. [Lester M. Morrison, M.D.and O. Arne Schjeide, Ph.D., Coronary Heart Disease and theMucopolysaccharides (Glycosaminoglycans) 12 (1974); Philip C. Champe andRichard A. Harvey, Lippincott's Illustrated Reviews: Biochemistry,148-50 (2^(nd) ed. 1994)]. One of ordinary skill in the art understandsthat chondroitin sulfate must have at least two, and potentially many,of these repeating units of glucuronic acid and sulfated galactosamine.

FIG. 1 provides a detailed description of the biosynthetic pathway forthe creation of GAGs, such as chondroitin sulfate. In addition, thepresent invention may include fragments of GAGs, such as fragments ofchondroitin sulfate. One of ordinary skill in the art at the time theinvention understands that “fragments of glycosaminoglycans” are groupsof saccharides that constitute less than two repeating units of theglycosaminoglycan. Hence, it is understood that fragments of thesesubstances would be composed of groups of saccharides that constitutefewer than two of the repeating units of the respective polymer.

For example, one of ordinary skill in the art understands that fragmentsof chondroitin sulfate are molecules composed of the saccharides thatcomprise the repeating units of chondroitin sulfate, but that arepresent in groups of less than the two repeating units described above.Thus, a molecule composed of a glucuronic acid and sulfatedgalactosamine would constitute a fragment of chondroitin sulfate.Indeed, there are eight different disaccharide structures that mayconstitute fragments of chondroitin sulfate. [Timothy E. Hardingham andAmanda J. Fosang, Proteoglycans: Many Forms and Many Functions, FASEBJ., 6:861-862 (1992)].

Other naturally occurring glycosaminoglycans may be used in thisinvention, for example, hyaluronic acid. Also, fragments of theglycosaminoglycans may also be utilized. A person of ordinary skill inthe art understands the terms “fragments of chondroitin,” “fragments ofchondroitin sulfate,” “fragments of chondroitin salts,” “fragments ofglycosaminoglycan” and “chondroitin sulfate fragments,” and furtherunderstands them to mean groups of saccharides (or salts thereof) thatconstitute less than two repeating units of the glycosaminoglycan.

One of skill would expect that fragments of chondroitin sulfate, forexample, would have the same utility as chondroitin sulfate itself.Chondroitin sulfate is broken down into smaller units within the body,and that it is reformulated in the production of cartilage and otherconnective tissue. Therefore, it is understood that the body utilizesfragments of chondroitin sulfate in the same manner as it utilizeschondroitin sulfate itself. The same is true with respect to “fragmentsof chondroitin,” “fragments of chondroitin salts,” and “fragments ofglycosaminoglycan.” Each of chondroitin, chondroitin salts and otherglycosaminoglycans, if ingested, is broken down by the body andreformulated in the production of cartilage and other connective tissue.Therefore, the body utilizes fragments of chondroitin in the same manneras it utilizes chondroitin itself, utilizes fragments of chondroitinsalts in the same manner as it utilizes chondroitin salts, and utilizesfragments of glycosaminoglycans in the same manner as it utilizesglycosaminoglycans.

Moreover, it is intended that the term GAG can extend to any compositionof matter that is insubstantially different from the GAGs asabove-described. An example of such a GAG-like compound that is withinthe scope of the present invention is pentosan polysulfate (PPS) as wellas salts thereof such as calcium-derived PPS and sodium PPS.Accordingly, a preferred GAG-like compound that may be used in thecompositions of the present invention is PPS.

PPS is a semi-synthetic polysulfated xylan that is a sulfated form of acompound extracted from beechwood hemicellulose consisting of repeatingunits of (1-4) linked β-D-xylano-pyranoses. More specifically, PPS isproduced by extracting these hemicellulose compounds via a series ofchemical reactions from the wood, and then adding numerous sulfategroups to the purified polysaccharide chains. This process results inlow molecular weight linear polysaccharide chains that carry numerousnegatively charged sulfate groups. PPS is a semi-synthetic heparinoidthat is considered an oversulfated form of a GAG.

There are several forms of PPS that display the above-describedactivities. Sodium PPS and a calcium-derived PPS (called CAPPS) may bothbe used to accomplish the functions of PPS. Each of these forms of PPSexhibit GAG-like activity, and will hereinafter be referred to asGAG-like compounds.

Pentosan's mechanism of action can be summarized as follows:

-   1. Anti-inflammatory activities through stabilization and    improvement of micro-circulation in the inflamed tissues and through    anti-Complement effects (decreases the release of the humoral    mediators of inflammation called the Complement cascade).-   2. Inhibition of chemotaxis of granulocytes, which are white blood    cells that contribute to inflammation.-   3. Stimulatory effect on proteoglycan synthesis.-   4. Stimulatory effects on hyaluronic acid synthesis by synovial    fibroblasts.-   5. Potent inhibition of catabolic enzymes including, human    granulocyte elastase (noncompetitive inhibition), hyaluronidase    (competitive inhibition), chondroitin-4-sulfatase and    N-acetyl-glucosaminidase at concentrations much more lower than that    of NSAIDs.    Other synthetic or semi-synthetic glycosaminoglycans or    glycosaminoglycan-like compounds, such as polysulfated    glycosaminoglycans, may be used in this invention.

Diacerein, a recently recognized organic compound found in plants of thegenus Cassia has anti-inflammatory effects through inhibition ofinterleukin-1B consequently collagenase production in articularcartilage is reduced. It reduces the fibrinolytic activity of synovialfibroblasts as well. It also dose-dependently inhibits chemotaxis(attraction of white blood cells) and superoxide anion production (thisis one of the “toxic oxygen species” or “free radicals”). These harmfulcompounds occur spontaneously in the body, especially during destructiveinflammation. Diacerein has analgesic and antipyretic activities. Itreduces the breakdown of chondroitin-4-sulfate resulting in an increasein the ratio of chondroitin-4-sulfate to chondroitin-6-sulfate. (Thisratio is pathologically decreased in degenerating cartilage.) It mildlyincreases prostaglandin synthesis, which allows it to have protectiveeffects on the gastric mucosa.

S-adenosylmethionine (SAMe) is an important endogenous compound, presentthroughout the body, and taking part in a great number of biologicreactions such as transsulfation reactions. In this role it is animportant reactant in the synthesis of many structural components ofconnective tissues, including proteins and proteoglycans. Thus, SAMe hassignificant anabolic effects which would enhance the actions of otheranabolic agents. SAMe also has anti-inflammatory effects by virtue ofits antioxidant action.

SAMe is compound synthesized in the body from adenosine triphosphate(“ATP”) and methionine (FIG. 2). It is present in many tissues,including the central nervous system. The primary CNS function of SAMeis to donate methyl groups in the reactions synthesizing various crucialcompounds, including neurotransmitters and phospholipids. For example,SAMe facilitates the conversion of phosphatidylethanolamine tophosphatidylcholine, which forms part of the inner, lipid layer of theplasma membrane. In so doing, SAMe increases membrane fluidity andenhances effectiveness of receptor/ligand binding. [Champe and Harvey,Biochemistry, 1994; Stramentinoli, G., “Pharmacologic Aspects ofS-Adenosylmethionine,” American J. Med., 83(5A):35 (1987); Baldessarini,F., “Neuropharmacology of S-Adenosyl Methionine,” American J. Med.,83(5A):95 (1987); Carney, M., “Neuropharmacology of S-AdenosylMethionine,” Clin. Neuropharmacol., 9(3):235 (1986); Janicak, P.,“S-Adenosylmethionine in Depression,” Alabama J. Med. Sci. 25(3):306(1988)]. These functions may also pertain to other methyl donors such asbetaine (trimethylglycine), 5-methyltetrahydrofolate, folic acid, anddimethylglycine. [Champe and Harvey, Biochemistry, 1994].

Superoxide dismutase is an enzyme present naturally in the tissues ofanimals and plants, which has recently been investigated as an agent inthe management of inflammation. It acts by intercepting toxic oxygenradicals in the intracellular space during destructive inflammatoryprocesses. It does not inhibit prostaglandin biosynthesis, but stops theoverproduction of prostaglandins resulting from destructiveinflammation. Some of its effects include inhibition of edema formationand inhibition of acute signs of inflammation and the secondaryarticular changes (stiffness and calcification) in adjuvant-inducedarthritis. Having no analgesic effects, it does not contribute to theoveruse of the affected joints that eventually leads to more damage ofthe articular cartilage, as NSAIDs can. Also, it has no adverse effectson the cardiovascular, central nervous or endocrine systems. FIG. 3provides a simplified diagram of the function of SOD.

L-ergothionine is an intracellular antioxidant naturally occurring inplants and animals, but not synthesized in human bodies: it comes onlyfrom dietary sources. The antioxidant properties of L-ergothioneinappear to be related to its ability to scavenge reactive oxygen species(free radicals), chelate various metallic cations, activate antioxidantenzymes such as glutathione peroxidase (SeGPx) and manganese superoxidedismutase (Mn SOD) and to inhibit superoxide-generating enzymes such asNADPH-Cytochrome C reductase, and to affect the oxidation of varioushemoproteins such as hemoglobin and myoglobin. Because all body tissuesdepend on these two oxygen carrier molecules, this characteristic isextremely beneficial. [Brummel, M. C., “In Search of a PhysiologicalFunction for L-ergothioneine,” Med. Hypotheses, 18(4):351-70 (December1985); Brummel, M. C., “In Search of a Physiological Function forL-ergothioneine, —II,” Med. Hypotheses, 30(1):39-48 (September 1989);Han, J. S., “Effects of Various Chemical Compounds on Spontaneous andHydrogen Peroxide-Induced Reversion in Strain TA104 of Salmonellatyphimurium,” Mutat. Res., 266(2):77-84 (April 1992); Arduini, A.,“Possible Mechanism of Inhibition of Nitrite-Induced Oxidation ofOxyhemoglobin by Ergothioneine and Uric Acid,” Arch. Biochem. Biophys.,294(2):398-402 (May 1992)].

Collagen Type II also has beneficial effects that help maintain thenormal balance between anabolism and catabolism. Specifically,connective tissue diseases may result from autoimmune processes, inwhich the immune system attacks and catabolizes the individual's ownconnective tissues as if it were a “foreign invader.” Oraladministration of collagen Type II can desensitize the immune system,preventing further attack and normalizing immune responses in theseindividuals. This decreases catabolic processes in the connectivetissues and maximize anabolism. Ingestion of collagen type II presentsthis molecule to the immune cells in the gut-associated lymphoid tissues(GALT, a.k.a., Peyer's patches). Interactions between the collagenmolecule and specific cells within the GALT activates mobile immunecells called T suppressor cells. These cells, in turn, moderate thedestructive immune reaction against the individual's own collagen typeII (in connective tissues).

Compounds in the tetracycline family include tetracycline, doxycycline,tetracycline analogs, and “tetracycline-like” compounds, and have beenused therapeutically for their anti-microbial effects. Current researchhas focused on “tetracycline-like” compounds which possess insignificantantimicrobial effects, but with anti-catabolic effects. Specifically,“tetracycline-like” compounds are polycyclic compounds that inhibittissue metalloproteinases which degrade extracellular matrix componentsincluding collagen and proteoglycans yet have insubstantialanti-microbial effects. This function of these compounds, as well asother compounds in the tetracycline family, may be related to theability of these compounds to chelate calcium and zinc ions. Forexample, doxycycline has been shown to inhibit collagenase activity inarticular cartilage.

Certain lipid extracts, termed nonsaponifiable, of avocado (genusPersea, especially P. americana) and the soybean (Glycine max) have alsobeen studied for their beneficial effects on connective tissues. Thesenonsaponifiable compounds are that part of the plant lipids that do notundergo saponification, i.e., they do not react with alkali to form asoap. There are many such compounds, and any particular avocado extractmay contain any number. Examples include fat soluble vitamins (A, D, E,and K), steroids such as phytoestrogens, sterols (bioflavonoids) andvolatile essentials oils (terpenes such as menthol, camphor, lycopene,gibberellic acid, limonene, cinnamaldehyde, carotenoids, and ubiquinone,also known as coenzyme Q.) [Mathews, C. K. & van Holde, K. E.Biochemistry, 2^(nd) ed., The Benjamin/Cummings Pub. Co., Inc., 1996, p.691.]

The avocado/soybean unsaponifiables (ASU) have been used in Europe underthe trade name Piascledine and have been used to treat osteoarthritisand other forms of arthritis [Thiers, M. H., “Unsaponifiableconstituents of avocado and soya oils. Treatment of certain forms ofarthralgia,” J. Med. Lyon 53(222):195-8 (February 1972) (article inFrench)], as well as soft-tissue inflammatory conditions [Trevoux, R.,“Unsaponifiable fractions of the avocado and soybean in gynecology,” J.Bynecol. Obstet. Biol. Reprod. 6(1):99-105 (January 1977) (article inFrench); Lamaud, M. E., et al., “Biochemical modifications of connectivetissue induced by the non-saponifiables of avocado and soy-bean oilsadministered percutaneously in the ‘hairless’ rat,” Pathol. Biol.26(5):269-74 (May-June 1978) (article in French)]. The mechanism ofaction of this compound is to stimulate chondrocyte expression of TGF(transforming growth factor) beta 1, TGF beta 2 and plasminogenactivator inhibitor 1 (“PAI-1”). By increasing PAI-1, ASU blocks thecascade that leads to metalloprotease activation [Boumediene K., et al.,“Avocado/soya unsaponifiables enhance the expression of transforminggrowth factor beta 1 and beta 2 in cultured articular chondrocytes,”Arthritis Rheum. 42(1): 148-56 (January 1999)]. ASU mixtures also reducethe spontaneous production of stromelysins, IL-6, IL-8 and prostaglandinE2 by chondrocytes. Additionally, ASUs decrease the effects of IL-1, andthereby reduce chondrocyte and synoviocyte production of collagenase.[Henrotin, Y. E., et al., “Effects of three avocado/soybeanunsaponifiable mixtures on metalloproteinases, cytokines andprostaglandin E2 production by human articular chondrocytes,” Clin.Rheumatol. 17(1): 31-9 (1998).]

TGF beta 1 and 2 are members of a family of homologouspolypeptidecytokines. These locally-acting hormones can have paracrineor autocrine effects and are made by a variety of cell types, includinglymphocytes, endothelial cells and macrophages. TGF beta has variedeffects in different tissues; it generally inhibits epithelial cellmetabolism. In connective tissues, however, it has been shown to be anindirect mitogen for fibroblasts and other cells of mesenchymal origin.It also can stimulate cellular production of fibronectin and collagen,and decrease protease activity, resulting in a net increase in matrixproduction. [Cotran, R. F., Kumar, V. and Robbins, S. L., Eds.,Pathologic Basis of Disease, 5^(th) ed., Saunders, 1994, pp. 40-42.]

Stromelysins are a subtype of proteinases that act on a variety ofextracellular matrix components, including proteoglycans, laminin,fibronectin, and collagen. Stromelysins are produced by fibroblasts,synoviocytes, and macrophages, among other cell types, under theinfluence of cytokines such interleukin-1 and tumor necrosis factoralpha. Interleukins and prostaglandins are among the many mediators ofinflammation. Reductions in levels of all of these compounds result in adecrease in pain and swelling, which are hallmarks of inflammation.

The fat-soluble vitamins present in ASU mixtures are necessary forgrowth, and augment the anabolic effects of TGF-beta. Because theystimulate TGF beta and also decrease degradative enzymes, as explainedabove, ASU mixtures can be said to have both anabolic and anti-cataboliceffects. Although some of the effects of ASUs overlap the effects ofother compounds in the present invention, ASUs contribute uniqueproperties to the group of compounds and provide very beneficial effectswhen used in combination with those other compounds. For example, whileglucosamine and ASUs both stimulate anabolic processes in connectivetissue cells, these compounds have different cellular mechanisms ofaction. Glucosamine acts in part through protein kinase C, while theeffect of ASUs, as stated above, is through transforming growth factor.Similarly, chondroitin and ASUs have inhibitory effects of IL-1. ASUs,however, inhibit the plasmin cascade, while chondroitin decreasesactivation of the complement cascade. Osteoarthritis is a complexdisease involving interplay of many cytokines at the cellular level.Because the different compounds of the present invention act ondifferent cytokines, they will have synergistic effects when used inappropriate combinations.

In placebo controlled, double blind trials, ASUs have been showneffective in reducing symptoms of osteoarthritis [Maheu, E., et al.,“Symptomatic efficacy of avocado/soybean unsaponifiables in thetreatment of osteoarthritis,” Arthritis Rheum. 41(1): 81-91 (January1998); Blotman, F., et al., “Efficacy and safety of avocado/soybeanunsaponifiables in the treatment of symptomatic osteoarthritis,” Rev.Rheum. Engl. Ed. 64(12): 825-34 (December 1997)] In these studies, sideeffects in the intervention groups were similar to those seen in theplacebo groups, indicating that ASUs are safe and well toleratedsubstances. ASUs as used in this invention can include any or allunsaponifiable lipids and/or combinations thereof. Examples ofcomponents of ASUs include but are not limited to: limonene, betacarotene, phyloquinone, and giberellic acid. As explained above, ASUscan include any of a number of classes of compounds including but notlimited to fat soluble vitamins, steroids, sterols and volatileessentials oils, or any combinations thereof. The invention includes,moreover, compositions which contain one avocado/soybean extract (ASU)or mixtures or combinations of such extracts (more than one ASU). Thereare many such combinations and all are intended to be included withinthe present invention.

The compounds of the present invention have several advantages overexisting therapies for connective tissue disorders, such as excellentsafety profiles. This is in part related to the fact that thesecompounds occur normally in the body and in various foods. Anothercharacteristic shared by the compounds is tendency for a slow onset ofaction. Pharmaceuticals, such as NSAIDs, tend to cause sudden changes inthe symptoms of disease. The endogenous compounds in the presentinvention work more slowly, by normalizing structures and functionswithin the body. While this action is beneficial, it does mean thatsymptoms will typically not be relieved immediately. For this reason, ananalgesic is included as an optional component of the compositions ofthe present invention. The analgesic is to be chosen from the group ofanalgesic compounds that have been shown to have minimal side effects attherapeutic doses, and also to have minimal negative effects onconnective tissue synthesis, as corticosteroid drugs and many NSAIDshave been shown to have. The analgesic that may be included in thecomposition of the present invention therefore is a nonsteroidalanalgesic that does not have anti-inflammatory effects. In other words,the analgesic is a nonsteroidal drug that is not an NSAID. Examples ofthe analgesics of the present invention include acetaminophen andtramadol. Except as discussed below, the preferred analgesic of thepresent invention is acetaminophen.

Acetaminophen is an analine derivative analgesic and antipyreticcompound working centrally through reversible inhibition of the enzymecyclo-oxygenase in the central nervous system. Acetaminophen also blocksthe peripheral pain impulse generation in nerve endings throughout thebody. It has been used extensively for symptomatic pain relief. Therelief of pain is beneficial for more than the obvious humane reasons.Since there are also important links between the emotional centers ofthe brain and the immune system, the relief of pain, and the resultingelevation in mood, has beneficial effects on inflammation and the manyother processes that are modulated by the immune system. Although itblocks cyclooxygenase activity, acetaminophen has very littleanti-inflammatory activity. Therefore, acetaminophen does not inhibitconnective tissue anabolism, as NSAIDs and corticosteroids do, andbecause it has minimal side effects at therapeutic doses, it is an idealanalgesic agent in the present invention. Another advantage to includinga safe analgesic in the present invention is that it will increase thelikelihood that patient compliance would be high, i.e., that patientswould continue taking the preparations long enough for disease-modifyingeffects to occur. Studies of isolated chondroprotective agents oftenhave a high rate of drop-out in the early weeks of therapy due topatient perception that the agent is not working. With the addition ofan analgesic, patients would be more inclined to continue therapy.

Cats are sensitive to acetaminophen because they do not metabolize iteffectively (poor hepatic conjugation with glucuronic acid andsubsequent depletion of glutathione occurs) [Goodman, A. and Goodman,L., The Pharmacological Basis of Therapeutics, 7^(th) ed., MacMillanPublishing Co., 1985, pp. 692-95; Ahrens, F., Pharmacology, Williams &Wilkins, 1996, pp. 174-75]. Accordingly, acetaminophen is notrecommended for use in cats.

The present invention comprises novel combinations of anabolic agents,anti-catabolic agents and antioxidant agents that maximize beneficial,anabolic effects (healing) and minimize any potential negative effects.In so doing, the present invention provides novel combinations of theseagents and anti-oxidant agents, for the protection, treatment and repairof connective tissues in humans and animals.

These compounds have a variety of beneficial effects on animal and humanconnective tissues, and, because they function via a variety ofmechanisms, work well in combination with each other. Although eachcompound has a number of functions, they can be roughly grouped as: (1)anabolic agents, including glucosamine, SAMe, AA, and ASUs, whichpromote growth processes in the body; (2) anti-catabolic agents, such aschondroitin sulfate, pentosan sulfate, collagen type II, tetracyclines,diacerin and ASUs, which inhibit destructive or catabolic processes; and(3) antioxidants, such as SOD, and L-ergothionine which prevent tissuedamage by scavenging toxic oxygen species (free radicals). Naturally,some compounds, such as ASUs, could be placed in more than one group, byvirtue of their overlapping functions. The present invention establishesthat combinations of these compounds would work well. In addition, ananalgesic could optionally be added to any of the individual compoundsrecited above or to a combination of them to provide relief from pain.Acetaminophen is the analgesic of choice because it does not havepowerful anti-inflammatory effects and therefore does not interfere withhealing of connective tissue. It also has minimal side effects attherapeutic doses, unlike NSAIDs which may cause gastrointestinalulceration or poor renal perfusion even at therapeutic doses. Thus, thepresent invention consists of various combinations of two or more of thefollowing agents: AA, glucosamine, chondroitin sulfate, pentosan,diacerin, S-adenosylmethionine, superoxide dismutase, L-ergothionein,collagen type II, tetracycline-like compounds, one or more ASUs and,optionally, one or more analgesics, e.g., acetaminophen. Examplesinclude, but are not limited to such combinations as: two anabolicagents (e.g., AA and glucosamine); an anabolic agent and ananti-catabolic agent (e.g., AA and pentosan); an anti-catabolic and anantioxidant (e.g., tetracyclicline and superoxide dismutase); orcombinations of more than two agents (e.g., glucosamine, SAMe and AA) orSAMe, ASUs, acetaminophen and diacerin. Examples of specific compoundsthat may be present in ASU extracts include but are not limited to:limonene, beta carotene, ubiquinone, and undecaprenol phosphate.

The following table shows possible combinations of pairs of thecompounds discussed above. The letter “X” marks novel combinations ofcompounds that form the novel compositions of the present invention. Theinvention also includes combinations of three or more agents of thefollowing compounds in the combinations shown on the table:

Glucosamine Chondroitin SAMe Pentosan Superoxide Dismutase (SOD)L-Ergothionine Collagen Type II Diacerin Arachadonic Acid

Tetracycline like compoundsOne or more avocado/soybean unsaponifiables.Analgesic, e.g., acetaminophenAs explained above, examples of desired combinations are marked by X.For example, the first X in the first row means a combination ofglucosamine and L-ergothionine or glucosamine and diacerin. Thecompositions of the present invention additionally comprise anyaggregation or addition of the combinations marked by X in any given rowor column. For example, the compositions disclosed in the first rowinclude combinations of glucosamine plus L-ergothionine plus diacerin,or glucosamine plus diacerin plus tetracycline-like compounds orglucosamine plus L-ergothionine plus diacerin plus AA plustetracycline-like compounds, and so on. Examples of compositionsdisclosed in the column designated “Collagen Type II” would includecombinations of collagen Type II plus SAMe plus pentosan, or collagenType II plus SAMe plus pentosan plus superoxide dismutase plusL-ergothionine, and so on. Examples of compositions disclosed in thecolumn designated “ASU” would include combinations of one or more ASUsplus glucosamine, or one or more ASUs plus SAMe plus pentosan, or one ormore ASUs plus collagen Type II plus SAMe plus pentosan plus superoxidedismutase plus L-ergothionine, and so on. Similarly, the table showsthat an analgesic, e.g., acetaminophen, can be combined with any othercompound listed in the table either singly or in any combination.

Superoxide Tetracycline Analgesic, e.g., Dismutase L- CollagenArachadonic like Acetamino- (SOD) Ergothionine Type II Diacerin Acidcompounds ASU phen Glucosamine X X X X X X Chondroitin X X X X X X SAMeX X X X X X X X Pentosan X X X X X X X X Superoxide X X X X X X XDismutase (SOD) L- X X X X X X Ergothionine Collagen X X X X X Type IIDiacerin X X X X Arachadonic X X X Acid Tetracycline X X like compoundsASU X

The present inventors have investigated certain combinations of theabove agents and have documented a novel response in severalcombinations. The effects of certain combinations of chondroitinsulfate, glucosamine, SAMe, arachidonic acid, collagen, pentosan, andsuperoxide dismutase were studied in cultures of adult bovine cartilagecells in different experiments (see example 2). Certain combinations hadan inhibitory effect (hypometabolic) in this particular study. Bothstimulatory and inhibitory novel interactions could be beneficial undervarious disease states. For example, a hypermetabolic state is part ofthe pathogenesis of some diseases. In such diseases, an inhibitory(hypometabolic) response would be beneficial to the individual. Futurestudies are planned to investigate the effects of a range ofconcentrations in the agents studied under various experimental models.Note that both increases and decreases in biosynthetic activity arenovel interactions and could be beneficial to organisms under selectedcircumstances. For example, many researchers currently believe thatosteoarthritis has a hypermetabolic component, especially in the earlystages of pathogenesis. Researchers are divided as to whether treatmentshould focus on agents that stimulate cartilage matrix production, oragents that are inhibitory and therefore make the cartilage environmentmore hypometabolic, which in turn could have a stabilizing effect on thecartilage tissue.

The compositions of the present invention may be administered via anyroute, including but not limited to intramuscularly, intravenously,orally, subcutaneously, rectally, topically, transcutaneously,intranasally, and intra-articularly, sublingually, intraperitoneally.Also, any salt of any of the present compounds may be used to aid inabsorption, e.g., glucosamine HCl, glucosamine sulfate, glucosaminephosphate, sodium chondroitin sulfate, calcium chondroitin sulfate,potassium chondroitin sulfate, etc. In addition, the composition can begiven in all common dosage forms including extended release dosageforms, pills, tablets, capsules, creams, liquids, aerosols, extendedrelease forms, injectables, etc.

The dosage ranges of the compositions of the present invention will varydepending upon the needs of the human or animal to which thecompositions are administered. The dosage ranges for the variouscomponents of the presently claimed compositions are as follows:

Compound Daily Dose Glucosamine Total dose range: 25 mg to 12 g Smallanimal: 25 mg-3 g. Human: 100 mg-4 g large animal: 300 mg-12 gChondroitin Total dose range: 15 mg-12 g sulfate Small animal: 15 mg-2 gHuman: 75 mg-4 g large animal: 300 mg-12 g SAMe Total dose range: 10mg-8 g Small animal: 10 mg-1 g Human: 75 mg-3 g large animal: 400 mg-8 gPentosan Total dose range: 3 mg to 3 g Small animal: 3 mg-1 g Human: 50mg-2 g large animal: 100 mg-3 g Superoxide Total dose range: 3 mg to 6 g(each mg dismutase containing >3000 McCord - Fridovich units) Smallanimal 3 mg-2 g Human: 5 mg-3 g large animal: 50 mg-6 g L-ergothioneineTotal dose range: 50 mg to 25 g Small animal: 50 mg-10 g Human: 50 mg-15g large animal: 100 mg-25 g Collagen Total dose range: 0.1 mg to 10 gType II Small animal: 0.1 mg-10 g Human: 0.1 mg-7.5 g large animal: 1.0mg. 10 g Diacerin Total dose range: 5 mg to 5 g Small animal: 5 mg-1 gHuman 20 mg-3 g large animal: 50 mg-5 g Arachadonic Total dose range: 10mg to 12 g acid Small animal: 10 mg-3 g Human: 10 mg-5 g large animal 50mg-12 g Tetracyclines Total dose range: 1.0 mg to 2 g Small animal: 1.0mg-1 g Human: 2 mg-1.5 g large animal: 50 mg.-2 g Avocado/soybean Totaldose range: 5 mg to 5 gram unsaponifiables Small animal: 5 mg to 1000 mgHuman: 50 mg to 1500 mg Large animal: 100 mg to 5 grams Or: 1 mg/kg to25 mg/kg Analgesic, e.g., Total dose range: 4 mg to 10 gramsacetaminophen Small animal (excluding cats): 4 mg to 1000 mg Human: 100mg to 4 gram Large animal: 100 mg to 10 gramsDoses are designed to cover the spectrum of body weights of smallanimals to large animals, with humans in the middle. The followingexamples are illustrative and do not in any way limit the presentinvention.

Example 1

In our preliminary investigations, surgical instability was induced inthe stifle joint of New Zealand white rabbits by modification of theHulth technique. Post-operatively, animals were exercised for 1 hourdaily. Experimental dietary formulas were evaluated for their cartilagestabilizing effect. The standard Harland (Teklad) rabbit diet (control);a standard diet also containing a 2% fungal oil containing 40% AA byweight (Arasco); and a standard diet containing also arachidonic acidand glucosamine/chondroitin were investigated. At 16 weeks, the medialfemoral condyles of all rabbits were removed and cartilage degenerationquantitatively evaluated with a modified Mankinhistological-histochemical grading system with safranin-O stainedslides. Cartilage from all joints with surgical instability exhibitedvarying degrees of macroscopic degenerative lesions. Our preliminaryresults indicated that adding arachidonic acid toglucosamine/chondroitin sulfate has the potential to produce a novelinteraction in cartilage. This novel interaction has the potential tohave a cartilage modulating effect.

Example 2 Procedure

Articular cartilage was resected from human or animal joints asepticallyand placed into a large petri dish in a small amount of DMEM/F-12 orF-12. The tissue was diced to 1-2 mm dimensions and transferred to asmall culture flask containing 20 mL DMEM or F-12+400 u/mL collagenase.The flask was placed on the shaker and incubated overnight.

The cell digest was repeatedly aspirated to increase release of cells.The cell digest was then placed into a 50 mL sterile centrifuge tube andcentrifuged in the Beckman at 1000 RPM for 10 minutes. The medium wasdiscarded by pipette and fresh DMEM/F-12 containing 1% FCS added.Depending on the size of the pellet, about 20-40 mL medium was added.Cell counts were determined by haemocytometer and the digest made up toa concentration of 100,000 cells/0.2 mL.

GAG Synthesis:

To conduct GAG synthesis, 0.2 mL was aliquoted into each well of a 96well plate using an 8 channel pipetter and the cells allowed to attachfor 24 hours. The media was removed and 0.3 mL of fresh 1% FCS mediaadded for 2-3 days. On the day of the experiment, the media was removedand the experimental solutions containing 35-sulfate isotope were added.The incubation was continued for 4 hours. Termination: at the end of theincubation period, the labeling media was removed, the cell layer wasrinsed repeatedly with cold 0.3 mL DMEM or F-12 (about 5×), and the celllayer was frozen for counting.

Counting of 96 Well Plates:

The cell layer for both the synthesis experiments were heated at 50degrees after adding 100 ul 1 N NaOH for a period of 2 hours. 200 ulscintillant was added and the plates were placed in the counter. Thedata was expressed as CPM/100,000 cells.

Indv. Agents: Agents Evaluation CPM/ Sum Combined Difference Agent100,000 cells (CPM) (CPM) (CPM) ChSO4-L 64 AA 70 134 18 −116 ChS04-H 50AA 70 120 81 −39 Glu-H 117 AA 70 187 16 −177 1% Sam 123 10Paleos 86 20962 −147 1% Sam 123 1Paleos 74 197 80 −117 3% Sam 42 1Paleos 74 116 100−16 3% Sam 42 10Paleos 86 128 83 −45 3% Sam 42 Collagen 118 160 90 −703% Sam 42 AA 70 112 104 −8 AA 70 10Pentos 76 146 106 −40 Collagen 7010Paleos 86 156 82 −74 Collagen 118 10Pentos 76 194 65 −129 Collagen 11810 Paleos 86 204 77 −127 ChSO4 = Chondroitin AA = Arachadonic Acid SAMe= S-adenosylmethionine Paleos = SOD Collagen = Collagen Pentos =Pentosan H = High concentration L = Low concentration

In this model, at the concentrations studied, the representativecombinations had an inhibitory (hypometabolic) effect in this particularstudy. This hypometabolic effect could be beneficial under variousdisease states, indeed both stimulatory and inhibitory novelinteractions could be beneficial under various disease states. Forexample, a hypermetabolic state is part of the pathogenesis of somediseases. In such diseases, an inhibitory (hypometabolic) response wouldbe beneficial to the individual. Future studies are planned toinvestigate the effects of a range of concentrations in the agentsstudied under various experimental models. Note that both increases anddecreases in biosynthetic activity are novel interactions and could bebeneficial to organisms under selected circumstances. For example, manyresearchers currently believe that osteoarthritis has a hypermetaboliccomponent, especially in the early stages of pathogenesis. Researchersare divided as to whether treatment should focus on agents thatstimulate cartilage matrix production, or agents that are inhibitory andtherefore make the cartilage environment more hypometabolic, which inturn could have a stabilizing effect on the cartilage tissue.

Example 3

A 4 year old child has juvenile rheumatoid arthritis in which the immunesystem inappropriately targets endogenous connective tissues withantibodies against native collagen type II. The resulting inflammationand degradation of cartilage causes pain and dysfunction in the synovialjoints. Present treatments include corticosteroids which non-selectivelysuppress the immune system, thus leaving the body vulnerable toinfectious disease, or methotrexate, which inhibits DNA synthesis,repair, and cellular replication, thus affecting not only the immunesystem but also intestinal mucosa, and the bone marrow. This child isgiven 2 mg of collagen type II daily, and SOD 10 mg daily. The collagendecreases the inappropriate immune attack, and the SOD inactivatesdestructive free radicals that damage cells. By preventing cellulardamage, the SOD helps maximize the normal function of joint tissuecells. This combination has no harmful side effects at therapeutic dosesand is a beneficial addition to existing therapies for rheumatoidarthritis.

Example 4

A 6 year old thoroughbred race horse has neutrophilic inflammation ofthe carpus. In this condition, trauma to the tissues of the jointinjures cells and therefore results in liberation of cytokines whichattract large numbers of neutrophils into the synovial space. Thisresponse is beneficial in cases of sepsis, but in non-septic conditionsthe neutrophils provide no useful service to the animal. Indeed, becauseneutrophils produce various degradative compounds, including superoxidemolecules, their presence in the joint contributes to a vicious cycle ofinflammation, tissue damage, and increased inflammation. Currently thiscondition is treated with nonsteroidal antiinflammatory drugs, whichsuppress prostaglandin synthesis and therefore have many side effects.This horse is given a mixture of diacerin 100 mg, pentosan 200 mg andSAMe, 1000 mg The diacerin and pentosan both inhibit chemotaxis (theattraction of white blood cells into the affected area) and thus reducethe numbers of neutrophils in the joint. Additionally, pentosanstimulates the synthesis of synovial fluid and thus supports normalfunction of the joint. Diacerin inhibits superoxide production; sincesuperoxide production is one of the mechanisms through which neutrophilshave their harmful effects, this action of diacerin is obviouslybeneficial. SAMe supports the structure and function of cell membranes,and therefore helps repair injured joint tissue cells thus blocking theevents that start the harmful inflammation. This combination has noharmful side effects at therapeutic doses and is a great improvementover existing therapies.

Example 5

A 47 year-old woman has severe knee osteoarthritis. Currently sherequires large doses of NSAIDs to control her symptoms. Although herorthopedic surgeon has recommended taking glucosamine/chondroitinsulfate, she has been reluctant to do so because these compounds areextracted from animal tissues and the patient is a strict vegetarian.Instead she takes diacerein 25 mg and ASU 250 mg, and 500 mg ofacetaminophen daily. The diacerin inhibits chemotaxis and therebyreduces inflammation in the knee joint. The ASU increases TGF beta 1 and2, stimulating repair of damaged joint tissues. The acetaminophen causesrapid analgesia, reducing the patient's symptoms without adverselyaffecting cartilage metabolism and without risk of gastrointestinalulceration. As a result of the reduction in pain, the patient decides toadd a 15 minute walk to her daily schedule. The controlled exercisefurther improves her physical and mental state.

Example 6

A 5 year old Jersey dairy cow is diagnosed with severe osteoarthritisfollowing an episode of fever and synovitis attributed to Lyme disease.This animal is the source of the owner's family milk supply and theowner wishes to treat the lameness with compounds that are “natural,”i.e., compounds that normally occur in plants and animal bodies, ratherthan pursuing more traditional solutions such as 1) culling the animal2) using non-steroidal anti-inflammatory drugs or 3) using steroids. Theanimal is treated with ASU 900 mg, SAMe 600 mg and glucosamine 500 mgdaily. This approach is an improvement over existing options for severalreasons. Because the compounds are natural components of plants andanimal bodies with documented wide margins of safety, there is lessconcern over metabolites secreted in the milk. Because the compounds areavailable orally, and are active in small amounts, they are easy toadminister to the animal in feed. The combined effect of the threecompounds is to reduce inflammation and pain, to support normalfunction, and to stimulate healing of connective tissues.

Based on the teaching of the present invention, one of skill in the artwould understand that combinations of the compounds taught by thepresent invention would act synergistically. For example, it isunderstood that glucosamine has stimulatory effects on chondrocytemetabolism which, by itself, aids in ameliorating diseases of cartilagedegradation. However, an increase in cell metabolism can also produce anincrease in free-radical production, as a natural by-product ofoxidative phosphorylation. The increase in free radical production woulddilute the beneficial effects of the glucosamine administration. Bycombining L-ergothioneine with glucosamine, one would expect an increasein metabolism and a reduction in free-radical damage, providing for agreater benefit than if compounds leading to one of these effects wereprovided. Therefore, one of skill in the art, based on the teaching ofthe present invention, would understand that combining glucosamine withL-ergothioneine would be more beneficial than providing either alone.The synergy that exists between certain compounds in the presentinvention also enables the use of lower doses of each compound. Althoughthese compounds are quite safe, there may be a potential for sideeffects. For example, large doses of glucosamine sulfate or chondroitinsulfate can cause gastrointestinal disturbances in some individuals. Inaddition, these compounds are costly; for these reasons, the ability tominimize the dose and still achieve beneficial effects is desirable.

Many modifications may be made without departing from the basic spiritof the present invention. Accordingly, it will be appreciated by thoseskilled in the art that within the scope of the appended claims, theinvention may be practiced other than has been specifically describedherein. Hence, the attached claims are intended to cover the inventionembodied in the claims and substantial equivalents thereto.

1. A composition comprising one or more avocado/soybean unsaponifiablesand a glycosaminoglycan.
 2. The composition according to claim 1,wherein the glycosaminoglycan comprises a natural, synthetic orsemi-synthetic glycosaminoglycan, a glycosaminoglycan-like compound, aglycosaminoglycan precursor or fragments of a glycosaminoglycan.
 3. Thecomposition according to claim 1, wherein the glycosaminoglycan has beenchemically modified by one or more of esterification, sulfation,polysulfation, acetylation and methylation.
 4. The composition accordingto claim 1, wherein the glycosaminoglycan is chondroitin or a saltthereof, hyaluronic acid, or a mixture of these.
 5. The compositionaccording to claim 1, wherein the glycosaminoglycan is chondroitinsulfate.
 6. The composition of claim 1, wherein a dose of the one ormore avocado/soybean unsaponifiables ranges from about 5 milligrams toabout 5 grams.
 7. The composition of claim 1, wherein a dose for a smallanimal of the one or more avocado/soybean unsaponifiables ranges fromabout 5 milligrams to about 1000 milligrams.
 8. The composition of claim1, wherein a dose for a human of the one or more avocado/soybeanunsaponifiables ranges from about 50 milligrams to about 1500milligrams.
 9. The composition of claim 1, wherein a dose for a largeanimal of the one more avocado/soybean unsaponifiables ranges from about100 milligrams to about 5 grams.
 10. The composition of claim 1, whereina dose of the one or more avocado/soybean unsaponifiables ranges fromabout 1 mg/kg to about 25 mg/kg.
 11. The composition of claim 1, whereina dose of the one or more avocado/soybean unsaponifiables for a smallanimal ranges from about 0.5 mg/kg to about 25 mg/kg.
 12. Thecomposition of claim 1, wherein a dose of the one or moreavocado/soybean unsaponifiables for a human ranges from about 0.5 mg/kgto about 25 mg/kg.
 13. The composition of claim 1, wherein a dose of theone or more avocado/soybean unsaponifiables for a large animal rangesfrom about 0.5 mg/kg to about 25 mg/kg.
 14. The composition of claim 1,wherein a dose of the glycosaminoglycan component ranges from about 15milligrams to about 12 grams.
 15. The composition of claim 1, wherein adose of the glycosaminoglycan component for a small animal ranges fromabout 15 milligrams to about 2 grams.
 16. The composition of claim 1,wherein a dose of the glycosaminoglycan component for a human rangesfrom about 75 milligrams to about 4 grams.
 17. The composition of claim1, wherein a dose of the glycosaminoglycan component for a large animalranges from about 300 milligrams to about 12 grams.
 18. The compositionof claim 1, wherein a dose of the glycosaminoglycan component for asmall animal ranges from about 1 mg/kg to about 75 mg/kg.
 19. Thecomposition of claim 1, wherein a dose of the glycosaminoglycancomponent for a human ranges from about 1 mg/kg to about 75 mg/kg. 20.The composition of claim 1, wherein a dose of the glycosaminoglycancomponent for a large animal ranges from about 1 mg/kg to about 75mg/kg.
 21. A method of preventing, treating or repairing damage toconnective tissue in humans and animals comprising administering acomposition according to claim 1 to a human or animal in need thereof.